CTCF is an evolutionarily conserved 11 Zinc finger (ZF) transcription factor with multiple DNA sequence specificity. CTCF-target sites are found in promoters of several genes including c-myc, Polo-like kinase and Pim-1 oncogenes. Normally, it negatively regulates cell proliferation. The CTCF gene is localized at the human chromosome locus 16q22.1 within a region that displays frequent cancer-associated deletions. Our mutational analysis of CTCF revealed several tumor-specific mutations resulting in amino acid substitutions at different positions critical either for ZF formation or for DNA base recognition. Each mutation selectively eliminates CTCF binding to some but not to all target DNA sites, suggesting that the oncogenic potential of mutant CTCF proteins may result from shifting the spectrum of target genes regulated by CTCF. These findings provide the first evidence for role of CTCF as a candidate tumor suppressor gene at 16q22. However, the most convincing experimental approach to demonstrate a CTCF-mediated cancer phenotype would be an observation of enhanced tumor susceptibility in CTCF deficient mice. Our preliminary results conclusively demonstrate that CTCF (+/-) knockout mice are predisposed to tumor development in multiple tissues compared to wild type littermates. Therefore, based on our experience with the p53 and p27 (Kip1) deficient mice, we plan to expand analyses of CTCF(+/-) mice to study in detail effects of CTCF haplo-insufficiency on spontaneous, radiation-, ENU-, DMBA-, and other carcinogens- induced tumor predisposition. There appears to be no tissue or a cell line negative for CTCF-containing RNA message(s). Homologous knockout of CTCF in transgenic mice results in early embryonic lethality. Therefore, complete loss of CTCF function in cancer is unlikely because it may be incompatible with cell proliferation. This hypothesis will be tested by analyzing mechanisms of inactivation of the remaining wild type CTCF allele (mutations and/or altered expression) in tumors developing from different tissues in irradiated or carcinogen- challenged CTCF (+/-) mice. In addition, we will test whether CTCF null mutation results in preimplantation lethality that may be expected if TCF has "cell-autonomous" essential, and universal function. We also anticipate genetic crosses to other tumor-prone mice such as p53 and p27 deficient mice to reveal genetic pathways, which cooperate with CTCF deficiency in normal development and tumorigenesis.